Skeletal muscle is comprised of multinucleated syncytia formed by the fusion of myoblasts. Through microarray studies, it was found that expression of C6ORF32 is upregulated early during human myoblast fusion, when myotubes contain between 2-5 myonuclei (Cerletti et al., 2006). Downregulation of C6ORF32 expression causes a block in myoblast fusion due to a significant decrease in myogenin expression (Yoon et al., 2007). By computational analyses, the predicted C6ORF32 protein contains only one domain of known function, a putative HDAC-binding domain. It was found that C6ORF32 binds to HDAC6, a class IIb HDAC known for its function in promoting cell motility. It is hypothesized that HDAC6 and C6ORF32 are functionally connected, and the interaction between HDAC6 and C6ORF32 positively regulates myogenic differentiation by reducing the de-acetylation activity of HDAC6. The regulatory connection between HDAC6 and C6ORF32 in skeletal muscle will be studied both in vitro and in vivo via the following specific aims: 1) Define the critical binding region of C6ORF32 to HDAC6, and demonstrate that binding of these two proteins occurs in vivo;2) Determine if HDAC6 regulates myoblast differentiation by modulating C6ORF32 acetylation/de-acetylation;3) Evaluate if C6ORF32 regulates myoblast differentiation by modulating HDAC6 activity;4) Determine if ablation of C6ORF32 expression in vivo yields to defects in muscle development, function and regeneration by affecting HDAC6 function. These studies will constitute the groundwork for understanding whether future pharmacological therapies targeting the regulatory interaction between C6ORF32 and HDAC6 are amenable to enhance myogenic repair. PUBLIC HEALTH RELEVANCE: C6ORF32 is an unnamed protein upregulated during fusion of human myoblasts. Previous studies in our laboratory have indicated that C6ORF32 might be involved in myogenic differentiation. More recent data indicates that C6ORF32 binds HDAC6, a critical HDAC known to regulate cell motility and cell-cell interactions. The current application will dissect the regulatory connection between HDAC6 and C6ORF32, and how this connection affects myogenic differentiation. This goal will be achieved through in vitro and in vivo studies. The proposed work will open the possibility of modulating the HDAC6-C6ORF32 regulatory interaction to enhance muscle cell fusion, with the broader scope of using C6ORF32 as a new target for muscle repair.